From x-ray crystallography to electron microscopy and back -- how best to exploit the continuum of structure-determination methods now available

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Ursula Barton
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1 From x-ray crystallography to electron microscopy and back -- how best to exploit the continuum of structure-determination methods now available Scripps EM course, November 14, 2007

2 What aspects of contemporary x-ray crystallography have made it a particularly powerful tool in structural biology? Molecular replacement: the body of pre-existing structural knowledge simplifies a new structure determination Density modification: elimination of noise by imposition of reality criteria in direct space Refinement: constraints enable you to incorporate chemical reality criteria

3 1. Phasing x-ray data from EM (TBSV; reovirus core) 2. Phasing electron diffraction data from coordinates derived from x-ray crystallography (aquaporin) 3. Docking an x-ray structure into an EM map (clathrin coat) 4. Lessons from x-ray crystallography for single-particle EM

4 X-ray crystallographic structure determination 1. Experimental phases map (modified map) build model Experimental phases are poor; density modification is useful whenever possible. Building rarely produces complete or fully correct model: model refine rephase rebuild and extend model refine (cycle) 2. MR phases map and MR model rebuild or extend model refine (cycle) Map is strongly biased, so it is much better to modify map based on solvent flattening or ncs, then continue with rebuilding and extending

5 Examples: phases from EM map as MR model, density modification from non-crystallographic symmetry (icosahedral: 5-fold in these two cases) TBSV: negative stain, 30 Å (1974) Reovirus: cryo, 30 Å (2000)

7 Protease σ3 σ1 μ1 σ3 μ1 σ1 σ2 λ1 λ2 Virion ISVP infectious or intermediate subviral particle Core Dryden, Baker et al. (1993).

8 Crystals of reovirus cores F432, a= 1255 Å Initial phases to 30 Å from modified EM density Phase extension by averaging

9 Averaging as basis for phase extension in x-ray crystallography Map Mask, average, and reconstitute SFs F s and ϕ s Works because true a.u. is smaller than crystallographic a.u., transform is effectively oversampled

10 Non-cryst. symmetry averaging and solvent flattening F,ϕ c F,ϕ FFT map F c,ϕ c dens. mod. FFT map'

12 Aquaporin-0 (AQP0): Molecular replacement with MOLREP, monomer as model Must refine unit cell (grid search) Refinement with CNS 1. Rigid body with unit-cell variation 2. Simulated annealing; rebuild from 2Fo-Fc with solvent flipping maps and SA omit maps to correct Gonen et al, 2004

13 Aquaporin-0 (AQP0): Gonen et al, 2005

14 Docking a model from x-ray crystallography (or NMR) into a cryoem density Two key resolution barriers: ~ 8-9 Å and ~ 4 Å Rigid-body refinement vs. more flexible refinement

15 Transferrin/TfReceptor Cheng et al (2004) Cell 116:

16 Molecular replacement: 1. Can a molecular model work as an initial reference for single-particle alignment, with appropriate filtering of spatial frequencies? 2. How can we best exploit molecular replacement in 2-D crystallography?

17 Clathrin coat 1. Density modification 2. ncs symmetry averaging Fotin et al, 2004

18 assembly Assembly - disassembly and of clathrin disassembly coats of clathrin coats uncoating cargo receptor vesicle formation adaptor clathrin

19 Anatomy of a clathrin coat C C proximal N knee distal ankle linker Clathrin lattice terminal domain N Triskelion = 3 x (Heavy Chain + Light Chain)

20 QuickTime and a Cinepak decompressor are needed to see this picture.

21 D6 barrel Musacchio slide here Musacchio, Smith, Grigorieff, Pearse, Kirchhausen

22 X-ray structure of clathrin fragments Proximal Region N-terminal Domain Ybe et al, 1999 terhaar et al, 1998

23 Comparison of EM and X-ray densities at 7.9 Å Top View Side View EM X-ray

24 Clathrin CHCR domain organization Proximal Region N-terminal Domain CHCR7 CHCR6 CHCR5 CHCR4 CHCR3 CHCR2 CHCR1 CHCR0 N-terminal Domain

25 Modeling structure of the whole leg CHCR7 CHCR6 CHCR5 CHCR4 CHCR3 CHCR2 CHCR1 CHCR0 N-terminal Domain

26 The helical tripod H CHCR7 CHCR6 CHCR5 CHCR4 CHCR3 CHCR2 CHCR1 CHCR0 N-terminal Domain

27 Two questions: 1. Can we improve a reconstruction by use of a model built into the density as reference? 2. Can we refine a model against the observed data (projected images)?

28 In crystallography, measured amplitudes are, by experimental arrangement, coming from an averaged structure. In single-particle EM, measured projections contain unique noise that will disturb estimate of projection parameters

29 X-ray: observations are amplitudes; refine model parameters against these observations, using chemistry as a constraint. If the model is incomplete, use refinement to improve phases, get better map, extend model. refine F.T. build Model Model Suitable map Model Refinement minimizes: F i calc (h;x) - F i obs (h) 2 R = F i obs (h) 2

30 EM: observations are projections; what parameters should be refined? Do we have enough power to refine against the following agreement factor? σ i calc (u,v;x,θ i ) - σ i obs (u,v) 2 R = σ i obs (u,v) 2 where σ i calc is the calculated projection, as a function of x, the model coordinates (and B s), and of θ i, the orientation and origin of the i th projection If not, what is a suitable compromise?

31 Would hope to have the following cycle: refine reconst build Model Model Suitable map Model

32 Karin Reinisch Tamir Gonen Yifan Cheng Piotr Sliz Alex Fotin Tom Walz Niko Grigorieff Tom Kirchhausen David DeRosier

33 X-ray: observations are amplitudes; refine model parameters against these observations, using chemistry as a constraint. If the model is incomplete, use refinement to improve phases, get better map, extend model. refine F.T. build Model Model Suitable map Model Refinement minimizes: F i calc (h;x) - F i obs (h) 2 R = F i obs (h) 2

Direct Method. Very few protein diffraction data meet the 2nd condition

$Direct Method. Very few protein diffraction data meet the 2nd condition$ Direct Method Two conditions: -atoms in the structure are equal-weighted -resolution of data are higher than the distance between the atoms in the structure Very few protein diffraction data meet the 2nd